An emerging paradigm for discovery in pharmaceutical and related biotechnology is the assembly of novel synthetic compound libraries by new methods of solid phase “combinatorial” synthesis. Combinatorial chemistry refers to a set of strategies for the parallel synthesis of multiple compounds or compounds mixtures, either in solution or on solid supports in the form of polymer-based resins (“beads”).
One implementation of combinatorial synthesis that is suitable to produce large chemical libraries relies on “one-bead-one-compound” (OBOC) libraries, which contain from 106 to 108 compounds. These libraries are screened by performing a variety of chemical and biochemical assays to identify individual compounds eliciting a response. The chemical identity of such specific compounds can then be determined by direct analysis, using, e.g., micro-sequencing and mass spectrometry.
In peptidic OBOC libraries, peptide sequences from a group of selected amino-acid building blocks are represented in an on-bead format in which many copies of only one sequence exist on each bead. OBOC libraries permit sifting through a list of peptide substrate sequences to correlate top hits with protein sequence databases. This strategy has been used successfully, for example to determine the optimal peptide substrates of peptide deformylase, a Fe2+ metalloenzyme that catalyzes N-terminal deformylation of nascent polypeptides in eubacteria (Hu et al., 1999, Biochemistry 38: 643-650).
Histone proteins serve to package DNA and to regulate its accessibility for processes including transcription, repair and replication. Six major histone classes are known. Two each of the class H2A, H2B, H3 and H4, assemble to form one nucleosome core particle around which DNA is wrapped. Acting as spools around which DNA winds, histones play a role in gene regulation. Histones achieve this control over DNA by acting as substrates to a host of posttranslational modifications that dictate function. Particularly dense with posttranslational information are the N-terminal histone tails. These can be covalently modified at several sites. Modifications of the histone tail include methylation, acetylation, phosphorylation, ubiquitination, sumoylation, citrullination, and ADP ribosylation. The core of the histones can also be modified. Combinations of modifications are thought to constitute a code, the so-called “histone code”. The histone code hypothesis asserts that histone-binding proteins and histone-modifying enzymes read and interpret the posttranslational states of properly “primed” histones to facilitate a particular outcome such as gene silencing, transcription, mitosis, etc. (Strahl et al., 2000, Nature 403: 41-45). However, thus far, the combinatorial complexity of the histone modification patterns has precluded a systematic inquiry of the patterns recognized by these “code readers”, proteins and enzymes that would display preferential specificity for these context-dependent modifications.
Protein deacetylases have been implicated in a variety of disease states including aging, diabetes, HIV regulation, cancer, cardiovascular disorders, and neurodegenerative diseases. Histone deacetylase Inhibitors are currently in clinical trials as cancer treatments. In particular, the Silent information regulator 2 (Sir2) family of NAD+ dependent protein deacetylases has been studied in recent years. This burgeoning interest can be attributed to the important roles of Sir2 enzymes (sirtuins) in regulating chromatin architecture, promoting transcriptional silencing and longevity, and in fatty acid metabolism. NAD+ dependent lysyl deacetylation is characterized by the stoichiometric release of nicotinamide and a novel metabolite, O-acetyl-ADP-ribose (OAADPr).
The Sir2 family of deacetylases is highly conserved among all forms of life with seven known human homologs (SIRT1-7). The most studied mammalian homolog, SIRT1, is a nuclear enzyme that has been found to deacetylate a number of proteins. Histones H3 and H4, p53, p300, TAF168, PCAF/MyoD, PGC-1alpha, FOXO1 and 4, NF-kappaB, and Tat are examples reported to be either biological targets and/or in vitro substrates of SIRT1. Collectively, the variety of proposed physiological targets reflects the functional diversity of SIRT1.
Identifying biological substrates is an important step in understanding the molecular basis for sirtuin phenotypes. However, in many of the studied cases, a certain degree of logical bias was used to link the target protein and SIRT1, as unbiased global substrate screening procedures were not used. Varying conclusions have been reached in regard to sirtuin substrate specificity and recognition. Most striking are the conclusions that sirtuins display minimal side-chain recognition (Avalos et al., 2002, Mol. Cell. 10: 523-535; Zhao et al., 2003, Structure (Camb) 11: 1403-1411) and that SIRT1 displays no substrate sequence specificity (Blander et al., 2005, J. Biol. Chem. 280: 9780-9785). In contrasting reports, clear substrate preferences were noted for yeast Sir2 and HST2 (Borra et al., 2004, Biochemistry 43: 9877-9887), and human SIRT2 (North et al., 2003, Mol. Cell. 11: 437-444).
To date, only one study has attempted to probe sirtuin substrate specificity using an acetyl-peptide library approach (Blander et al., 2005). Curiously, the study reported that SIRT1 displayed no substrate specificity in vitro, a conclusion based on an oriented peptide library. With this method, only globally preferred amino-acids could be resolved, and the actual sequence of individual peptides was not elucidated. Although the peptide library technique has been successful for examining protein kinase substrate specificity (Songyang at al., 1994, Curr. Biol. 4: 973-982), its usefulness toward protein deacetylases remains uncertain.